Transcriptional profiling of Scedosporium apiospermum enzymatic antioxidant gene battery unravels the involvement of thioredoxin reductases against chemical and phagocytic cells oxidative stress

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Transcriptional profiling of Scedosporium apiospermum enzymatic antioxidant gene battery unravels the involvement of thioredoxin reductases against chemical

and phagocytic cells oxidative stress

Cindy Staerck, Julie Tabiasco, Charlotte Godon, Yves Delneste, Jean-Philippe Bouchara, Maxime Fleury

To cite this version:

Cindy Staerck, Julie Tabiasco, Charlotte Godon, Yves Delneste, Jean-Philippe Bouchara, et al.. Tran-

scriptional profiling of Scedosporium apiospermum enzymatic antioxidant gene battery unravels the

involvement of thioredoxin reductases against chemical and phagocytic cells oxidative stress. Medical

Mycology, Oxford University Press, 2019, 57, pp.363-373. �10.1093/mmy/myy033�. �inserm-01814253�

doi: 10.1093/mmy/myy033 Advance Access Publication Date: 0 2018 Original Article

Original Article

Transcriptional profiling of Scedosporium apiospermum enzymatic antioxidant gene battery unravels the involvement of thioredoxin reductases against chemical and phagocytic cells oxidative stress

Cindy Staerck

, Julie Tabiasco

, Charlotte Godon

, Yves Delneste

, Jean-Philippe Bouchara

and Maxime J.J. Fleury

1

Groupe d’Etude des Interactions H ˆote-Pathog `ene (EA 3142), UNIV Angers, UNIV Brest, Universit ´e Bretagne- Loire, Angers, France,

CRCINA, INSERM, Universit ´e de Nantes, Universit ´e d’Angers, Angers, France,

Laboratoire d’Immunologie et Allergologie, Centre Hospitalier Universitaire d’Angers, France and

Laboratoire de Parasitologie- Mycologie, Centre Hospitalier Universitaire, Angers, France

∗

To whom correspondence should be addressed. Maxime J.J. Fleury, Pharm.D., PhD, Groupe d’Etude des Interactions H ˆote-Pathog `ene, EA 3142, Institut de Biologie en Sant ´e-IRIS, CHU, 4 rue Larrey, 49933 Angers, France. Tel: + 33 02 44 68 83 68;

E-mail: maxime.fleury@univ-angers.fr

J.P.B. and M.J.J.F. are members of the ECMM/ISHAM (European Confederation of Medical Mycology/

International Society for Human and Animal Mycology) working group Fungal respiratory infections in Cystic Fibrosis (Fri-CF).

Received 23 January 2018; Revised 22 March 2018; Accepted 1 May 2018; Editorial Decision 5 April 2018

Abstract

Scedosporium species rank the second, after Aspergillus fumigatus, among the filamentous fungi coloniz- ing the airways of patients with cystic fibrosis (CF). Development of microorganisms in the respiratory tract depends on their capacity to evade killing by the host immune system, particularly through the oxidative response of macrophages and neutrophils, with the release of reactive oxygen species (ROS) and reactive nitrogen species (RNS). This is particularly true in the airways of CF patients which display an exacerbated inflammatory reaction. To protect themselves, pathogens have developed various enzymatic antioxidant systems implicated in ROS degradation, including superoxide dismutases, catalases, cytochrome C peroxi- dases, chloroperoxidases and enzymes of the glutathione and thioredoxin systems, or in RNS degradation, that is, flavohemoglobins, nitrate reductases, and nitrite reductases. Here we investigated the transcriptional regulation of the enzymatic antioxidant gene battery in 24-h-old hyphae of Scedosporium apiospermum in response to oxidative stress induced chemically or by exposure to activated phagocytic cells. We showed that 21 out of the 33 genes potentially implicated in the oxidative or nitrosative stress response were over- expressed upon exposure of the fungus to various chemical oxidants, while they were only 13 in co-cultures with macrophages or neutrophils. Among them, genes encoding two thioredoxin reductases and to a lesser extent, a peroxiredoxin and one catalase were found to be overexpressed after chemical oxidative stress as well as in co-cultures. These results suggest that thioredoxin reductases, which are known to be virulence factors in other pathogenic fungi, play a key role in pathogenesis of scedosporiosis, and may be new drug targets.

Key words: Scedosporium apiospermum, ROS, RNS, oxidative stress, enzymatic antioxidant, thioredoxin reductase.

Introduction

Although saprophytes usually live in highly polluted soils and contaminated water, Scedosporium species are opportunistic

pathogens that cause in receptive hosts various diseases rang- ing from localized infections such as subcutaneous myce- tomas to severe and often fatal disseminated infections in

CThe Author(s) 2018. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. 1

immunocompromised individuals.

About 10 species are rec- ognized today in the Scedosporium genus,

but the prominent species remain the closely related S. boydii and S. apiospermum in Europe,

and S. aurantiacum in Australia.

With a prevalence ranging from 2 to 17.4%, Scedospo- rium species rank the second, after Aspergillus fumigatus, among the filamentous fungi colonizing the respiratory tract of patients with cystic fibrosis (CF).

These fungi, now considered as prominent pathogens in the CF context, are usually responsible for a chronic colonization of the air- ways.

However, they may also cause true respiratory infections, similar to those due to A. fumigatus like bron- chitis and allergic bronchopulmonary mycoses,

as well as severe and often fatal disseminated infections following lung transplantation.

To colonize the respiratory tract, fungi must adhere to the host tissues and acquire the extracellular iron necessary for nu- merous essential physiological processes like respiration and er- gosterol synthesis, but they also need to cope the host immune response. Macrophages and neutrophils are major effector cells of the innate immune system, releasing various antimicrobial compounds during the inflammatory reaction triggered by the pathogens, including reactive oxygen species (ROS) as superox- ide anions and hydrogen peroxide, and reactive nitrogen species (RNS) as nitric oxide.

This is particularly true in the CF lung, which is characterized by an exacerbated inflammatory reaction.

To prevent macromolecules degradation during oxidative and nitrosative stresses, pathogens have developed a variety of enzymatic antioxidants, including superoxide dismutases (SODs), catalases, cytochrome c peroxidases, chloroperoxidases, enzymes of the glutathione or thioredoxin systems, flavohe- moglobins, nitrate and nitrite reductases.

Prior to the sequencing of S. apiospermum,

few pathogenic factors have been identified in Scedosporium species. Studies in this field focused on proteolytic enzymes thought to be in- volved in the host tissue invasion

or in protection of the fungus against the host immune defenses. In S. boydii, a cytoso- lic Cu/Zn-SOD called SODC

and three distinct catalases have been identified, and one of them, that is, catalase A1 (CatA1), was purified and characterized.

Genes encoding SODC and CatA1 were sequenced, and it was demonstrated that CatA1 gene was highly expressed in response to the oxidative stress.

Recently, a genome-wide analysis in S. apiospermum permitted to identify 31 other genes encoding proteins putatively impli- cated in ROS or RNS detoxification.

In order to get a bet- ter knowledge of the pathogenic mechanisms of Scedosporium species, we investigated here the transcriptional response of these genes to chemical oxidative stresses and in co-culture with acti- vated phagocytic cells.

Methods

Fungal strain and growth conditions

Study was conducted on the reference strain Scedosporium apiospermum IHEM 14462 originally isolated from a sputum sample from a CF patient and previously used for whole genome sequencing.

The fungus was maintained by weekly passages on potatoe-dextrose-agar (PDA, containing in g/L: dextrose, 20;

infusion from potatoes, 200; and bacteriological agar, 15; CON- DALAB, Madrid, Spain) supplemented with chloramphenicol 0.5%, with incubation at 37

C. Conidia from 9-day-old cul- tures collected by scraping as previously described

were inocu- lated in yeast extract-peptone-dextrose (YPD; containing in g/L:

yeast extract, 10; peptone, 20; glucose, 20; and chloramphenicol 0.5%, pH 7) broth in Petri dishes (30 mL per dish; 2 × 10

coni- dia per mL), which were incubated for 24 h at 37

C to obtain hyphae.

For some experiments, the fungus was also cultivated under environmental conditions encountered in the CF airways. In this aim, the fungus was cultivated in YPD broth supplemented with 90 mM NaCl or buffered at pH 5.3 by the addition of 0.5 M 2- (N-morpholino)ethanesulfonic acid with incubation under nor- mal air conditions, or in YPD broth with incubation under 5%

CO

. After 24 h of incubation, hyphae were recovered by cen- trifugation (4000 g, 15 min) and stored at − 80

C before total RNA extraction. All experiments were performed in triplicate and hyphae grown in YPD broth under normal air conditions were used as control.

Chemical induction of an oxidative or nitrosative stress

For these experiments, 24-h-old hyphae were incubated for 3 h at 37

C in the presence of various chemicals (from Sigma Aldrich, Saint-Quentin Fallavier, France) at a concentration ranging from 31.25 μM to 4 mM: (i) menadione (single addition at T0) or hy- drogen peroxide (addition at T0 and then every hour), which induce a general stress; (ii) diamide or cumene hydroperox- ide (single addition at T0), which generate an oxidative stress triggering specifically the glutathione and thioredoxin systems;

and (iii) DETA NO (single addition at T0), which releases NO and thus generates a nitrosative stress. In sum, 24 h-old hyphae grown in YPD broth under normal air conditions and incubated for 3 h without any oxidant were used as control. After 3 h of incubation, hyphae were recovered and stored at −80

C before total RNA extraction.

Co-cultivation with phagocytic cells

Co-culture experiments were realized as previously described

with slight modifications. The human myelomonocytic leukemia

cell line THP-1 (ATCC) was cultivated in Roswell Park Memo- rial Institute (RPMI) 1640 medium, and the human promyelo- cytic cell line HL60 (ATCC) in IMDM. Both culture media were supplemented by the addition of fetal calf serum (10%; PAA Laboratories, Pashing, Austria), L-glutamine (2 mM), sodium pyruvate (1 mM), nonessential amino acids (0.1 mM), HEPES (10 mM), and antibiotics (penicillin 100 U/ml and streptomycin 100 mg/mL, both from Lonza, Verviers, Belgium). Cell cultures were realized in T175 vented flasks at 37

C under 5% CO

with a density of 2 × 10

to 1 × 10

cells/ml, according to the recom- mendation of ATCC. Phorbol 12-myristate 13-acetate 50 ng/ml (Sigma Aldrich) was added to induce differentiation of THP-1 cells into macrophages (also referred as THP-1 macrophages).

Dimethylsulfoxide (DMSO; Sigma Aldrich) 1.3% was used to induce differentiation of HL60 cells into neutrophils (also re- ferred to as HL60 neutrophils). After differentiation, the cells were washed and incubated overnight in complete medium at a concentration of 1 × 10

cells per cm

. Before co-cultures, the cells were activated by the addition of 2 mg/ml lipopolysac- charides from Escherichia coli O111:B4 (LPS; Sigma Aldrich).

Co-cultures were performed at a ratio of one cell for three 24- h-old fungal hyphae in T175 flasks for 1 to 24 h of incubation for LPS-activated TPH-1 macrophages, and 15 to 100 min for LPS-activated HL60 neutrophils. Controls were realized with 24-h-old hyphae in RPMI medium with LPS but without phago- cytic cells for each incubation time. The absence of changes in gene expression level in co-cultures with nonactivated phagocytic cells was also verified. After co-culture, hyphae were recovered and stored at − 80

C. Biological triplicates were realized for each experiment.

RNA extraction and reverse transcription

For RNA extraction, hyphae were ground in liquid nitrogen, and extraction was performed on 100 mg of the homogenate with Nucleospin RNA plant kit (Macherey Nagel, D ¨uren, Germany).

Genomic DNA was then digested with DNase. Total RNA was quantified by fluorimetry on a qubit fluorimeter 2.0 (Invitrogen, Cergy Pontoise, France) and integrity was verified by 1% agarose gel electrophoresis.

Three micrograms of RNA were then reverse transcribed with the high capacity complementary DNA (cDNA) reverse tran- scription kit (Applied Biosystems, Waltham, MA, USA) accord- ing to the manufacturer’s instructions. Reverse transcription was performed on thermal cycler Bioer Genepro, for 5 min at 25

C, followed by 2 h at 37

C and 5 min at 85

C. The cDNA were then 1/15th diluted in water and stored at − 20

C before analysis.

Gene expression analysis

The expression of the 33 target genes was analyzed by real time (RT)- quantitative polymerase chain reaction (qPCR) on a Step

One Plus thermalcycler (Applied Biosystems Foster City, CA, USA). RT-qPCR was performed in a 12.5-μl reaction volume containing 1 × Fast SYBR

Green Master Mix (Applied Biosys- tem), 0.4 μM of each primer (Supplementary Table 1), and 20 ng of cDNA. The PCR thermal profile consisted in an initial incuba- tion at 95

C for 20 s, followed by 40 cycles of 30 s at 95

C, 30 s at 60

C, and 15 s of denaturation at 95

C with a final extension of 1 min at 60

C. Melting curves were obtained at the end of PCR, by increasing temperature from 60

C to 95

C at 0.3

C/s, to identify possible primer dimerization or off-target amplifica- tion. For each gene, RT-qPCR was done in duplicate for each of the three biological replicates. Genes encoding actin and glycer- aldehyde 3-phosphate dehydrogenase (GAPDH) were used for normalization. Relative quantification (RQ) of gene expression was determined from the threshold cycle (Ct) values, according to the following formula

(Livak and Schmittgen 2001):

Ct

= Ct

−Ct

, corresponding to experimental conditions

Ct

= Ct

− Ct

, corresponding to control conditions

RQ = 2

(

)

Statistical analysis

RT-qPCR data were compared using the Two-ANOVA test, and results were considered significant when P < .05.

Results

Cultivation in CF conditions has little effect on the expression of target genes

Gene expression was evaluated in three CF conditions (90 mM

NaCl, acid pH, or 5% CO

). Only a few genes were

overexpressed in these conditions. Two genes encoding a

thioredoxin and a flavohemoglobin (SAPIO CDS8608 and

SAPIO CDS1359, respectively) were overexpressed in the

presence of 90 mM NaCl, with 3.2- and 3.1-fold changes

in expression levels, respectively. Five genes were slightly

overexpressed at pH 5.3, encoding respectively a catalase per-

oxidase, a peroxiredoxin, a thioredoxin, a thioredoxin reduc-

tase, and a nitrite reductase (SAPIO CDS10583, SaPrx2, SA-

PIO CDS8608, SAPIO CDS10274, and SAPIO CDS10292,

respectively) with 2.8-, 4.7-, 4.2-, 6.8- and 4.3-fold changes in

expression levels, respectively. Likewise, five genes were overex-

pressed under 5% CO

, three of them being overexpressed also

at pH 5.3 (SAPIO CDS10583, RQ = 3.4; SAPIO CDS8608,

RQ = 2.9; SAPIO CDS10292, RQ = 5.5). The last two genes encoding a Cu/Zn-SOD (SAPIO CDS7433) and a Mn-SOD (SA- PIO CDS3426) were overexpressed only under 5% CO

with a 5.1- and 5.0-fold increase in the expression level, respectively.

A single gene was overexpressed in these three conditions, SA- PIO CDS8608, encoding thioredoxin (Supplemental Fig. 1).

Chemical oxidants strongly influence the expression of some enzymatic antioxidant genes

Gene expression was evaluated in hyphae stressed by mena- dione, a superoxide anion inductor, at concentrations rang- ing from 0.03125 to 2 mM to induce a global oxida- tive stress. And 13 out of the 33 genes studied were over- expressed (Fig. 1, Table 1). SAPIO CDS1830 encoding a thioredoxin reductase was the most overexpressed, followed by SAPIO CDS0416, SAPIO CDS6039, SAPIO CDS4327, and SAPIO CDS8864 encoding a glutaredoxin, a glu- tathione reductase, a Cu/Zn-SOD, and a glutathione perox- idase, respectively. Although the maximum expression level was reached for low concentrations of menadione (0.25 or 0.5 mM) for some genes, in most cases the gene expression levels progressively increased with the concentration of menadione up to the highest concentration tested.

Exposure to hydrogen peroxide, that also induces a global oxidative stress, resulted in the overexpression of 12 out of the 33 target genes (Supplemental Fig. 2, Table 1). Eight of these genes were also overexpressed in response to menadione, in- cluding the abovementioned genes. SAPIO CDS4931 encoding a glutathione reductase was the most overexpressed, followed two other genes encoding proteins of the glutathione system (SAPIO CDS6039 and SAPIO CDS0416) and four of the genes encoding proteins of the thioredoxin system (SAPIO CDS9937, SAPIO CDS10274, SaPrx2 and SAPIO CDS1830).

Diamide and cumene hydroperoxide, which specifically target proteins of the glutathione and thioredoxin systems, induced the overexpression of five of the 10 genes of the glutathione system and three of the six genes of the thioredoxin system for diamide (Supplemental Fig. 3A, Table 1), and of three and four genes of the glutathione and thioredoxin systems for cumene hydroperoxide (Supplemental Fig. 3B, Table 1). The highest fold changes were obtained for SAPIO CDS10274 with diamide and SAPIO CDS6039 with hydroperoxide cumene.

Finally, the use of DETA NO, that leads specifically to a nitrosative stress, induced the overexpression of two of the tar- get genes, a flavohemoglobin (SAPIO CDS8682) and a nitrite reductase (SAPIO CDS10292) (Fig. 2, Table 1).

Genes of the thioredoxin system are strongly induced by co-cultivation with phagocytic cells

To better understand the potential role of enzymatical antioxi- dant gene battery in an innate immune context, the gene expres-

sion profile was also analyzed in 24-h-old hyphae co-cultivated with LPS-activated TPH-1 macrophages or HL60 neutrophils.

No changes were seen in the gene expression levels after co- culture with nonactivated THP-1 macrophages or HL60 neu- trophils classically used as control in such experiments since they produce ROS and RNS at a very low basal level.

Only four genes showed an overexpression in co-culture with activated TPH-1 macrophages. After 3 hours of co-culture, a maximum expression level was observed for SAPIO CDS1830 gene, encoding one of the thioredoxin reductases, (3.6-fold change compared to the control). The three others genes, SAPIO CDS10274 (encoding another thioredoxin reductase), SaPrx2 (encoding a peroxiredoxin), and SAPIO CDS4185 (en- coding one of the catalases) displayed a maximum level expres- sion at 12 h of co-culture, with a 14.6-, 13.0-, and 9.4-fold increase, respectively (Fig. 3, Table 1). Expression of the other target genes remained unchanged.

Shorter times of incubation were used with LPS-activated HL60 neutrophils due to the quick release of ROS after their activation. In co-culture with these cells, 13 genes were over- expressed (Fig. 4, Table 1). For most of them (11), the maxi- mum expression level was reached very quickly, after 30 min of co-culture whereas a 60 or 75 min of incubation was needed for the two other genes, that is, SAPIO CDS8682 and SAPIO CDS1359 genes, which encode flavohemoglobins.

The highest relative expression levels were obtained for two genes of the thioredoxin system, that is, SAPIO CDS10274 and SaPrx2, which encode a thioredoxin reductase and a peroxire- doxin (11- and 10-fold changes, respectively), followed by a SAPIO CDS4185 and SAPIO CDS4327, which encode a cata- lase and a Cu/Zn-SOD (8.4- and 8.3-fold changes, respectively;

Fig. 4, Table 1).

To sum up, Figure 5 illustrates the genes significantly overex- pressed in response to a general stress induced chemically (mena- dione or hydrogen peroxide) or generated by phagocytic cells.

And 19 genes were found to be overexpressed in at least one condition, 10 of them being other expressed in two conditions (exposure to menadione and to hydrogen peroxide or to LPS- activated HL60 neutrophils) and four of them in three condi- tions (SAPIO-CDS4327, SAPIO CDS6474, SAPIO-CDS4185, and SAPIO-CDS10274 encoding a Cu/Zn-SOD, a Mn-SOD, a catalase and a thioredoxin reductase, respectively). Interestingly, only two genes encoding proteins of the thioredoxin system (SA- PIO CDS1830 and SaPrx2) were overexpressed in the four con- ditions.

Discussion

Few data are available today regarding the pathogenesis of Sce-

dosporium infections. To establish an infection, pathogens must

evade the host immune defenses. One prominent mechanism

in the host defense against pathogens is the oxidative burst

Figure 1.Relative gene expression levels in 24-h-oldS. apiospermumhyphae after induction of oxidative stress by menadione. Coding sequences (CDS) are indicated in Genbank database with the prefix SAPIO for genome ofS. apiospermumstrain IHEM 14462 except forSaPrx2, which not considered as a coding sequence in the draft genome sequence²⁸and was identified by a blastn analysis of the whole genome. Data were compared using the Two-ANOVA test (^∗∗∗∗P

<.0001;^∗∗∗.01<P<.001;^∗∗.05<P<.01;^∗P<.05). This Figure is reproduced in color in the online version ofMedical Mycology.

Table 1. Gene expression levels in 24-h-old S. apiospermum hyphae exposed to a chemical oxidative stress or co-cultivated with LPS- activated phagocytes.

Exposure to Co-culture with LPS-activated

Gene annotation and Menadione H

O

Diamide Cumene DETA NO THP-1 macrophages HL60 neutrophils

accession number Cu/Zn-SODs

CDS2117

17.4 (2) 5.3 (2) / / / 2.2 (3 h) 2.7 (30 min)

CDS3212

2.8 (2) 4.2 (1) / / / 1.5 (12 h) 1.9 (30 min)

CDS4327

19.7 (1) 10.5 (2) / / / 3.5 (12 h) 8.3 (30 min)

CDS7433

9.1 (0.25) 5.1 (2) / / / 1.3 (7 h) 1.4 (30 min)

Mn-SODs

CDS3426

2.4 (2) 1.1 (2) / / / 1.7 (3 h) 1.9 (30 min)

CDS6474

15.6 (0.25) 8.6 (2) / / / 2.0 (7 h) 4.4 (30 min)

CDS8679

4.2 (2) 3.8 (0.25) / / / 1.8 (3 h) 2.2 (30 min)

Catalases

CDS2912

6.0 (2) 9.4 (2) / / / 2.0 (24 h) 2.2 (30 min)

CDS4185

11 (2) 5.6 (2) / / / 9.4 (12 h) 8.4 (30 min)

Catalases-peroxidases

CDS4198

4.6 (2) 2.7 (1) / / / 1.2 (7 h) 2.6 (30 min)

CDS10583

11.9 (2) 3.5 (2) / / / 2.4 (3 h) 3.8 (30 min)

Glutathione peroxidases

CDS4353

6.0 (0.5) 6.5 (2) 7.5 (2) 14.1 (4) / 1.4 (12 h) 1.6 (60 min)

CDS8864

17.8 (0.25) 9.6 (2) 4.9 (1) 72.1 (2) / 1.5 (12 h) 1.5 (30 min)

Glutaredoxins

CDS0416

31.1 (0.5) 28.6 (0.25) 15.3 (1) 10.2 (1) / 1.2 (12 h) 1.9 (30 min)

CDS3187

3.6 (2) 1.8 (2) 2.6 (1) 5.2 (1) / 1.8 (3 h) 1.7 (30 min)

CDS3482

2.8 (2) 1.7 (0.25) 4.0 (4) 6.4 (2) / 2.2 (1 h) 2.5 (30 min)

CDS8578

3.6 (0.5) 2.8 (2) 6.2 (4) 27.0 (4) / 1.9 (3 h) 2.6 (30 min)

CDS3162

3.3 (2) 4.7 (0.25) 7.3 (1) 6.2 (1) / 2.0 (1 h) 1.4 (30 min)

Glutathione reductases

CDS4534

1.8 (1) 6.4 (0.25) 4.4 (1) 2.4 (2) / 1.7 (1 h) 1.6 (60 min)

CDS4931

9.4 (0.5) 79.4 (0.5) 22.8 (1) 26.5 (2) / 2.1 (12 h) 7.7 (30 min)

CDS6039

21.6 (0.5) 34 (2) 13.0 (1) 85.0 (2) / 1.9 (24 h) 2.0 (30 min)

Peroxiredoxins

CDS4142

4.9 (2) 5.9 (2) 3.2 (2) 12.0 (2) / 1.7 (12 h) 1.7 (60 min)

CDS9937

11.3 (0.5) 28.7 (2) 13.8 (1) 33.6 (2) / 1.3 (12 h) 1.2 (75 min)

14.0 (0.5) 20 (0.5) 22.9 (1) 25.6 (2) / 13.0 (12 h) 10.0 (30 min) Thioredoxin

CDS8608

6.7 (0.5) 4.5 (2) 7.7 (1) 10.1 (4) / 2.1 (3 h) 2.7 (30 min)

Thioredoxin reductases

CDS1830

68.5 (2) 14.9 (0.25) 4.1 (4) 66.3 (4) / 3.6 (3 h) 3.9 (30 min)

CDS10274

8.1 (1) 20.7 (0.5) 77.2 (1) 40.0 (2) / 14.6 (12 h) 11 (30 min) Cytochrome C peroxidase

CDS3675

2.1 (2) 1.3 (0.125) / / / 1.6 (3 h) 1.9 (30 min)

Chloride peroxidase

CDS8967

10.7 (2) 5.2 (2) / / / 2.8 (12 h) 5.4 (30 min)

Flavohemoglobins

CDS1359

1.8 (2) 14.1 (0.25) / / 6.1 (4) 2.6 (3 h) 3.7 (75 min)

CDS8682

1.9 (0.03125) 1.4 (0.25) / / 18.4 (4) 2.5 (12 h) 3.5 (60 min)

Nitrate reductase

CDS10293

8.4 (2) 1.7 (0.25) / / 6.2 (4) 2.7 (3 h) 4.5 (30 min)

Nitrite reductase

CDS10292

13.1 (2) 6.8 (2) / / 54.4 (2) 2.6 (24 h) 7.4 (30 min)

Coding sequences (CDS) are indicated in Genbank database with the prefixSAPIO for genome ofS. apiospermumstrain IHEM 14462 except for SaPrx2, which was not considered as a coding sequence in the draft genome sequence²⁸,and was identified by a blastn analysis of the whole genome. Data correspond to the maximum expression levels (expressed in fold changes) for each gene and in parenthesis is indicated the concentration (in mM) of the chemical oxidant or the duration of co-culture needed to reach this value. Significant increases in the gene expression levels are highlighted in bold font. /: not determined.

Figure 2.Relative gene expression levels in 24-h-oldS. apiospermumhyphae after induction of nitrosative stress by DETA NO. Coding sequences (CDS) are indicated in Genbank database with the prefix SAPIO for genome of S. apiospermumstrain IHEM 14462.²⁸Data were compared using the Two- ANOVA test (^∗∗∗∗P<.0001;^∗∗∗.01<P<.001). This Figure is reproduced in color in the online version ofMedical Mycology.

Figure 3.Relative gene expression levels in 24-h-oldS. apiospermumhy- phae co-cultivated with LPS-activated THP-1 macrophages. Coding sequences (CDS) are indicated in Genbank database with the prefix SAPIO for genome of S. apiospermumstrain IHEM 14462 except forSaPrx2, which was not consid- ered as a coding sequence in the draft genome sequence ofS. apiospermum, and was identified by a blastn analysis of the whole genome.²⁸This Figure is reproduced in color in the online version ofMedical Mycology.

response of phagocytic cells and the subsequent release of ROS and RNS. The role of ROS in the host defense against pathogens is particularly well illustrated by the occurrence of infections in patients with chronic granulomatous disease (CGD). Indeed, mutations of Nox gene encoding the NADPH oxidase, which characterize this genetic disease, result in a defective produc- tion of superoxide anions by phagocytic cells, correlated with an increased susceptibility to respiratory pathogens.

Interest- ingly, most of the pathogens causing respiratory infections in CGD patients are also common CF pathogens, including Staphy-

lococcus aureus and Burkholderia cepacia for bacteria, and A. fumigatus, Scedosporium, and Lomentospora species, the Rasamsonia argillacea species complex or Exophiala dermati- tidis for filamentous fungi.

Evasion of pathogens to the oxidative or nitrosative stress is essential to survive in the environment and to cause an infec- tion. Regarding S. apiospermum, recent analysis of the genome revealed a set of 33 genes encoding SODs (7), catalases (4), proteins of the glutathione system (10), and of the thioredoxin system (6), chloroperoxidase (1), cytochrome c peroxidase (1), flavohemoglobins (2), nitrate reductase (1), and nitrite reductase (1), which are potentially implicated in protection of the fungus against the oxidative or nitrosative stress.

Here we showed overexpression of 21 of these genes in response to an oxidative or nitrosative stress induced chemically or by co-cultivation of the fungus with phagocytic cells, suggesting for these genes a role in the protection of the fungus against the host immune defenses.

Twelve of these genes were not significantly overexpressed in none of our experimental conditions, including genes encoding two Cu/Zn-SODs. Nevertheless, one cannot disregard the in- volvement of these genes in protection of the fungus against the host defenses. First, they may be constitutively expressed as the Cu/Zn-SOD of Candida glabrata.

The functional redundancy in the same protein family, as well as between some protein fam- ilies also should be considered. For instance, expression of one of the four catalases identified in S. apiospermum was unchanged.

This catalase (encoded by SAPIO CDS4198) is a bifunctional catalase also degrading peroxide as glutathione peroxidases and peroxiredoxins, and several genes encoding these last enzymes were found to be overexpressed. Likewise, for each protein fam- ily, at least one of the encoding genes was found to be overex- pressed, perhaps compensating the lack of response of the others.

Moreover, the study was conducted on 24-h-old hyphae, and one may speculate the expression of some genes specifically in other morphological stages of the fungus. For example, expression of SAPIO CDS3212 encoding a Cu/Zn-SOD was unchanged in all of our experimental conditions. However, we showed previously that this enzyme is a glycosyl phosphatidyl inositol-anchored cell wall protein expressed exclusively at the surface of the conidia which are the infecting form of the fungus.

Likewise, four SOD genes were identified in A. fumigatus genome, two of them were highly expressed in conidia while AfSOD3 was expressed only in hyphae.

Finally, since macrophages are less involved in the im- mune response against hyphae than neutrophils, our model could explain that a higher number of genes were modulated when 24- h-old hyphae of S. apiospermum were exposed to neutrophils (13 genes overexpressed vs. only four with macrophages).

Twenty-one genes were significantly overexpressed in at least

one of our experimental conditions, and 12 upon exposure to

both a chemically induced oxidative stress and an oxidative

stress induced either by THP-1 or HL60 cells. Interestingly,

Figure 4.Relative gene expression levels in 24-h-oldS. apiospermumhyphae co-cultivated with LPS-activated HL60 neutrophils Coding sequences (CDS) are indicated in Genbank database with the prefix SAPIO for genome ofS. apiospermumstrain IHEM 14462 except forSaPrx2, which not considered as a coding sequence in the draft genome sequence ofS. apiospermum,and was identified by a blastn analysis of the whole genome.²⁸This Figure is reproduced in color in the online version ofMedical Mycology.

while some genes encoding proteins of the glutathione system were highly overexpressed in response to a chemically induced oxidative stress (the two genes encoding glutathione peroxidases, one of the five genes encoding a glutaredoxin, and two of the three genes encoding glutathione reductases), only one of them (SAPIO CDS4931 encoding a glutathione reductase) was found to be overexpressed in co-culture with LPS-activated HL60 neu- trophils. In C. albicans, the glutathione reductase GLR1 is also

induced upon exposure to hydrogen peroxide

as well as in co-

culture with human neutrophils

and was shown to be involved

in virulence of the yeast in the Galleria mellonella model.

Like-

wise, this enzyme is essential to virulence of C. neoformans in

mice.

Further studies are needed to define the role of this glu-

tathione reductase in the pathogenesis of scedosporiosis. As for

the other genes of the glutathione system, their lack of response

upon exposure to phagocytic cells suggests a prominent role for

Figure 5.Venn representation of the genes significantly overexpressed in response to a general stress induced chemically (menadione or hydrogen peroxide) or generated by phagocytic cells. Accession numbers of the cod- ing sequences (preceded in the Genbank database by the prefix SAPIO CDS) that were significantly overexpressed in the different experimental conditions studied, are indicated. Only two CDS (in red and bold font) were overexpressed in each of the four conditions illustrated here.SaPrx2, which was not consid- ered as a coding sequence in the draft genome sequence ofS. apiospermum strain IHEM 14462,²⁸was identified by a blastn analysis of the whole genome.

In dark blue are indicated the Genbank accession number ofS. apiospermum CDS encoding superoxide dismutases, in pink CDS encoding catalases, in red CDS encoding proteins of the glutathione system, in purple CDS encoding proteins of the thioredoxin system, in green CDS encoding peroxidases, and in brown CDS encoding proteins involved in NO degradation (the color figure is available in the online version of the manuscript). This Figure is reproduced in color in the online version ofMedical Mycology.

other antioxidant genes, such as components of the thioredoxin system.

Only two genes encoding a thioredoxin reductase (SAPIO CDS1830) and a peroxiredoxin (SaPrx2) were overexpressed upon exposure to both menadione and hydro- gen peroxide as well as in co-culture with THP1 and HL60 cells, whereas two other genes were upregulated in these co-cultures as well as upon exposure to either menadione or hydrogen per- oxide, that is, SAPIO CDS4185 and SAPIO CDS10274, which encodes one catalase and the other thioredoxin reductase. These genes were also highly expressed in response to diamide and cumene hydroperoxide, particularly the thioredoxin reductase gene SAPIO CDS10274, which showed the highest changes in expression level.

Catalases are known to detoxify hydrogen peroxide and to participate to evasion of the pathogen to the oxidative stress.

Nevertheless, in most cases, they did not seem to be essential for virulence. For example, three catalases are described in A.

fumigatus, including the spore-specific CatA and two mycelial catalases, the monofunctional catalase Cat1 and the catalase- peroxidase Cat2, but the delta CatA mutant was as virulent as the wild-type strain in a rat model of aspergillosis and the double mutant delta cat1 delta cat2 exhibited only delayed infection in this experimental model.

Likewise, four catalase genes have been described in C. neoformans, and no differences were seen in virulence in a mouse model between a wild-type strain and single

or even quadruple mutants.

Conversely, a single catalase gene CAT1 was identified in C. albicans, and a markedly decreased virulence was reported for the homozygous null mutant in a mouse model of disseminated candidiasis.

The role of fungal peroxiredoxins in virulence is poorly doc- umented. In A. fumigatus, the Asp f3 allergen was identified as a member of the peroxiredoxin protein family, and disruption of the encoding gene resulted in a hypersensitivity to external superoxide and in a total lack of virulence in mice, suggesting that these enzymes could be therapeutic targets.

Our results also suggest that genes encoding the two thiore- doxin reductases in S. apiospermum could play an important role in protecting the fungal cells against ROS and RNS. There is now an increasing body of evidence that these enzymes display a high potential for drug design and vaccine development.

For example, the C. albicans thioredoxin reductase (CaTrxR or TRR1) is highly overexpressed under oxidative stress and is implicated in ROS and RNS degradation, especially in re- sponse to neutrophils,

as well as in virulence in a mouse model of candidiasis.

Likewise, recombinant CaTrxR protein induced high levels of serum specific antibodies and reduced the fungal burden in experimental disseminated candidiasis in mice.

In addition, recent studies on the thioredoxin reductase inhibitor auranofin

highlighted its promising antimicrobial po- tential against various bacteria,

as well as some pathogenic yeasts

and filamentous fungi.

Finally, our experiments also showed overexpression of the flavohemoglobin gene SAPIO CDS8682 and the nitrite reduc- tase gene SAPIO CDS10292 upon exposure to DETA-NO. Al- though further investigations on these genes are needed, recent studies performed on A. fumigatus which revealed a prominent role of the flavohemoglobin FhpA in protection of the conidia against RNS, also showed no differences in killing of the conidia by macrophages and in virulence for mice between the wild-type strain and the FhpA mutant.

In conclusion, this study provides new insights into the re- sponse of S. apiospermum to the oxidative or nitrosative stress.

As observed in other human pathogenic fungi, several genes were deregulated in S. apiospermum hyphae upon exposure to ROS and RNS, suggesting a role in protecting the fungus. Further studies are needed to confirm their involvement in protection of the fungus against the host immune defenses. Considering liter- ature data, a particular attention should be paid to genes encod- ing some proteins of the thioredoxin system, particularly SaPrx2 and the thioredoxin reductase genes. These genes which play an essential role in virulence in other fungal pathogens, were over- expressed in most of our experimental conditions, and therefore could be considered as potential targets for drug development.

Supplementary material

Supplementary data are available at MMYCOL online.

Acknowledgments

The financial support of

for Cindy Staerck’s PhD thesis is gratefully acknowledged (RF20140501104).

Declaration of interest

The authors report no conflicts of interest. The authors alone are respon- sible for the content and writing of this paper.

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